Chir99021

Mouse ESCs were obtained as previously reported
³⁹ (link),
⁴⁰ (link),
⁴¹ (link) and maintained in 2i/L medium (1000 IU/mL LIF [Millipore, ESG1107], 1 μM PD0325901 [Miltenyi Biotec, 130–103‐923] and 3 μM CHIR99021 [Miltenyi Biotec, 130–103‐926] in N2B27 basal medium) on fibronectin‐coated (Millipore, FC010) plates. N2B27 basal medium contained one volume of DMEM/F12 (Gibco, 11,320–033) combined with one volume of neurobasal medium (Gibco, 21,103–049) supplemented with 0.5% N2 supplement (Gibco, 17,502–048), 1% B27 supplement (Gibco, 17,504–044), 2 mM GlutaMAX‐l (Gibco, 35,050–061), 1% MEM NEAA (Gibco, 11,140–050), 1% penicillin/streptomycin (Gibco, 15,140–122), 50 mg/L bovine serum albumin (Gibco, 15,260–037) and 110 μM β‐mercaptoethanol (Sigma, M3148).

iPSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA (Life Technologies, 0.5mM). All cell cultures were maintained at 37°C and 5% carbon dioxide. MN differentiation was performed using an adapted version of a previously published protocol (Hall et al., 2017). Briefly, iPSCs were first differentiated from neuroepithelium by plating to 100% confluency in chemically defined medium consisting of DMEM/F12 Glutamax, Neurobasal, L- Glutamine, N2 supplement, non-essential amino acids, B27 supplement, β-mercaptoethanol (all from Life Technologies) and insulin (Sigma). Treatment with small molecules from day 0 to day 7 was as follows: 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience), and 3 μM CHIR99021 (Miltenyi Biotec). On day 8, the neuroepithelial layer was enzymatically dissociated using dispase (GIBCO, 1 mg/mL), plated onto laminin coated plates and next patterned for 7 days with 0.5 μM retinoic acid and 1μM Purmorphamine. At day 14, MN precursors were treated with 0.1μM Purmorphamine for a further 4 days before being terminally differentiated in 0.1 μM Compound E (Enzo Life Sciences) to promote cell cycle exit.

hiPSCs were maintained using standard protocols and were differentiated into astrocytes as described previously, generating highly enriched (>90%) populations of astrocytes (Supplementary Figure S1A) (8 (link),11 (link),48–51 (link)). hiPSCs were maintained on Geltrex (Life Technologies) with serum-free Essential 8 Medium media (Life Technologies), and passaged using EDTA. After neural conversion (7 days in a chemically defined medium containing 1 μM Dorsomorphin (Millipore), 2 μM SB431542 (Tocris Bioscience) and 3.3 μM CHIR99021 (Miltenyi Biotec), neural precursors were patterned for 7 days with 0.5 μM retinoic acid and 1 μM purmorphamine, followed by a 4-day treatment with 0.1 μM purmorphamine. After a propagation phase (60–120 days) with 10 ng/ml FGF-2 (Peprotech), astrocytes were terminally differentiated in presence of BMP4 (10 ng/ml, R&D) and LIF (10 ng/ml, Sigma-Aldrich) for 30 days. Informed consent was obtained from all patients and healthy controls in this study. Experimental protocols were all carried out according to approved regulations and guidelines by UCLH’s National Hospital for Neurology and Neurosurgery and UCL’s Institute of Neurology joint research ethics committee (09/0272).

We seeded 40,000 INS-1E (AddexBio, San Diego, CA, USA) per cm² (flasks) and cultivated these cells in medium containing RPMI 1640 with glutamine (Life Technologies, Carlsbad, CA, USA), 1 mmol/l sodium pyruvate (Life Technologies), 50 μmol/l β-mercaptoethanol (Life Technologies), 10 mmol/l HEPES (Life Technologies), 10% fetal bovine serum (FBS) (Biochrom, Berlin, Germany), 100 IU/ml penicillin and 100 μg/ml streptomycin (Life Technologies). After 4 days, INS-1E were detached using 0.05% trypsin (Life Technologies), seeded at 100,000 cells per cm² (24- or 96-well plate) and cultured in the aforementioned medium for another 3 days. Then, cells were fasted for FBS for 4h and treated with 0.1, 1 or 5 μg/ml recombinant Sfrp5 (R&D Systems, Wiesbaden, Germany) for 24h. INS-1E cells were also incubated with 0.2 ng/ml interleukin (IL)-1β (R&D Systems) as positive control for the inhibition of cell viability. Treatment of the cells with 10 μmol/l CHIR99021 (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) served as positive control of an activated canonical Wnt signalling pathway.

Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM β-mercaptoethanol (ThermoFisher) and 1 mM sodium pyruvate (ThermoFisher). Naive medium (t2iLGoY)^{19 (link)}: 50:50 mixture of DMEM/F12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l-glutamine (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng ml⁻¹ human leukaemia inhibitory factor (made in-house), 250 μM l-ascorbic acid (Sigma), 10 μg ml⁻¹ recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), 10 μM Y-27632 (Abcam). Primed hiPS cell medium (KSR/FGF2): DMEM/F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng ml⁻¹ recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher). Primed hiPS cell medium (Essential 8 (E8)): 10 ml of E8 supplement (Gibco) to 500 ml medium basal (Gibco), supplemented with 1% penicillin-streptomycin (Gibco).

Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM β-mercaptoethanol (ThermoFisher) and 1 mM sodium pyruvate (ThermoFisher). Naive medium (t2iLGoY)^{19 (link)}: 50:50 mixture of DMEM/F12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l-glutamine (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng ml⁻¹ human leukaemia inhibitory factor (made in-house), 250 μM l-ascorbic acid (Sigma), 10 μg ml⁻¹ recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), 10 μM Y-27632 (Abcam). Primed hiPS cell medium (KSR/FGF2): DMEM/F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng ml⁻¹ recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher). Primed hiPS cell medium (Essential 8 (E8)): 10 ml of E8 supplement (Gibco) to 500 ml medium basal (Gibco), supplemented with 1% penicillin-streptomycin (Gibco).